Endocannabinoid signaling as an intrinsic component of the circuits mediating adaptive responses to repeated stress exposure in adult male sprague dawley rats.

Evidence implicates the endocannabinoid (eCB) system as a negative modulator of neural and endocrine responses to acute stressors. Recently, eCB signaling was also reported to contribute to habituation of hypothalamo-pituitary-adrenal (HPA) axis responses to repeated homotypic stress. The present studies were initiated to distinguish a potential role of eCB signaling in the expression vs. the acquisition of habituation of the HPA axis response to repeated stress. In each of three experiments, adult male Sprague Dawley rats were exposed to daily, 30-minute sessions of loud white noise (95 dB), which resulted in a progressive decrease in HPA axis response over successive days. Cannabinoid receptor 1 (CB1) antagonist AM251 (0.5, 1.0 or 2.0 mg/kg, i.p.) was used to examine the role of eCB signaling in homotypic stressor habituation and heterotypic (novel) stressor cross-sensitization of neuroendocrine activity. Pretreatment with high dose (2.0 mg/kg) AM251 before each of 7 consecutive, daily loud noise exposures (acquisition of habituation) resulted in potentiation of stress-induced HPA axis activation and disruption of habituation. After an 8th loud noise exposure without AM251 pretreatment, the same group of rats displayed a habituated plasma corticosterone (CORT) level similar to that of controls, indicating that CB1 receptor antagonist pretreatments did not disrupt the acquisition of habituation. In two additional experiments, rats acquired habituation to loud noise drug free, then lower doses of AM251 (0.5 and 1.0 mg.kg) were administered before a final exposure (expression of habituation) to the homotypic stressor and/or a novel heterotypic stressor. CB1 receptor antagonism disrupted the expression of CORT response habituation and some of the c-fos mRNA reduction associated with it and facilitated novel stressor sensitization in doses that did not potentiate acute responses to these stressors. Collectively, these data suggest a progressive intensification of neural eCB signaling at CB1 receptors with repeated stress exposures.

Prefrontal Cortex KCa2 Channels Regulate mGlu5-Dependent Plasticity and Extinction of Alcohol-Seeking Behavior.

Identifying novel treatments that facilitate extinction learning could enhance cue-exposure therapy and reduce high relapse rates in alcoholics. Activation of mGlu5 receptors in the infralimbic prefrontal cortex (IL-PFC) facilitates learning during extinction of cue-conditioned alcohol-seeking behavior. Small-conductance calcium-activated potassium (KCa2) channels have also been implicated in extinction learning of fear memories, and mGlu5 receptor activation can reduce KCa2 channel function. Using a combination of electrophysiological, pharmacological, and behavioral approaches, this study examined KCa2 channels as a novel target to facilitate extinction of alcohol-seeking behavior in rats. This study also explored related neuronal and synaptic mechanisms within the IL-PFC that underlie mGlu5-dependent enhancement of extinction learning. Using whole-cell patch-clamp electrophysiology, activation of mGlu5 in ex vivo slices significantly reduced KCa2 channel currents in layer V IL-PFC pyramidal neurons, confirming functional downregulation of KCa2 channel activity by mGlu5 receptors. Additionally, positive modulation of KCa2 channels prevented mGlu5 receptor-dependent facilitation of long-term potentiation in the IL-PFC. Systemic and intra-IL-PFC treatment with apamin (KCa2 channel allosteric inhibitor) significantly enhanced extinction of alcohol-seeking behavior across multiple extinction sessions, an effect that persisted for 3 weeks, but was not observed after apamin microinfusions into the prelimbic PFC. Positive modulation of IL-PFC KCa2 channels significantly attenuated mGlu5-dependent facilitation of alcohol cue-conditioned extinction learning. These data suggest that mGlu5-dependent facilitation of extinction learning and synaptic plasticity in the IL-PFC involves functional inhibition of KCa2 channels. Moreover, these findings demonstrate that KCa2 channels are a novel target to facilitate long-lasting extinction of alcohol-seeking behavior.SIGNIFICANCE STATEMENT Alcohol use disorder is a chronic relapsing disorder that is associated with compulsive alcohol-seeking behavior. One of the main causes of alcohol relapse is the craving caused by environmental cues that are associated with alcohol. These cues are formed by normal learning and memory principles, and the understanding of the brain mechanisms that help form these associations can lead to the development of drugs and/or behavior therapies that reduce the impact that these cues have on relapse in alcoholics.

In vivo monitoring of rat brain endocannabinoids using solid-phase microextraction.

Aim: Solid-phase microextraction is proposed to measure concentrations of anandamide and 2-arachidonoyl glycerol in live rat brains in response to stress. Materials & methods: Solid-phase microextraction fibers were prepared from steel with 1.5 mm extraction coating. 24 male rats were divided into groups based on brain region, stria terminalis or posterior hypothalamus and loud noise or control groups. The fibers were desorbed in acetonitrile-water (75:25) and analyzed by ultraperformance LC-MS/MS. The linear range of the method was 0.05-50 ng/ml and the in vivo concentrations were found to be between 0.3 and 40 ng/ml. Conclusion: The new approach was successfully used to determine the concentrations of anandamide and 2-arachidonoyl glycerol in vivo and could be used in the future to measure other endogenous compounds.

Effects of alcohol exposure during development on play behavior and c-Fos expression in response to play behavior.

Developmental exposure to alcohol can produce characteristic physiological and cognitive deficits, often termed Fetal Alcohol Spectrum Disorder (FASD). More recently, social deficits have been shown to occur both in FASD and animal models of FASD; the behavioral and neural bases of these deficits remain to be determined. It was hypothesized that changes in sensory processing may in part underlie the social deficits seen in FASD. This study used a rat model of FASD and social play, a behavior critical to adult social functioning, to begin to examine this hypothesis. Somatosensory cues from dorsal contact to the nape of the neck, critical to the initiation of pinning, were systematically degraded by administration of different doses of xylocaine, a topical anesthetic. Neuronal activity after 1h of play was assessed by measurement of c-Fos immunoreactivity (IR) in different brain regions. Ethanol-exposed rats showed an increased frequency of pinning during social play and were more sensitive to the degradation of somatosensory cues compared to the control groups, suggesting difficulties in processing somatosensory cues. Neuronal activity in the somatosensory cortex induced by play was significantly decreased in the ethanol-exposed group compared to the non-treated group. The c-Fos IR in the nucleus accumbens was altered in a sexually dimorphic manner in the ethanol-exposed group. Thus, the behavioral and brain measures are consistent with the hypothesis that ethanol exposure during development induces alterations in social play via deficits in processing somatosensory cues that are important to social play.

Adolescent caffeine consumption increases adulthood anxiety-related behavior and modifies neuroendocrine signaling.

Caffeine is a commonly used psychoactive substance and consumption by children and adolescents continues to rise. Here, we examine the lasting effects of adolescent caffeine consumption on anxiety-related behaviors and several neuroendocrine measures in adulthood. Adolescent male Sprague-Dawley rats consumed caffeine (0.3g/L) for 28 consecutive days from postnatal day 28 (P28) to P55. Age-matched control rats consumed water. Behavioral testing for anxiety-related behavior began in adulthood (P62) 7 days after removal of caffeine. Adolescent caffeine consumption enhanced anxiety-related behavior in an open field, social interaction test, and elevated plus maze. Similar caffeine consumption in adult rats did not alter anxiety-related behavior after caffeine removal. Characterization of neuroendocrine measures was next assessed to determine whether the changes in anxiety were associated with modifications in the HPA axis. Blood plasma levels of corticosterone (CORT) were assessed throughout the caffeine consumption procedure in adolescent rats. Adolescent caffeine consumption elevated plasma CORT 24h after initiation of caffeine consumption that normalized over the course of the 28-day consumption procedure. CORT levels were also elevated 24h after caffeine removal and remained elevated for 7 days. Despite elevated basal CORT in adult rats that consumed caffeine during adolescence, the adrenocorticotropic hormone (ACTH) and CORT response to placement on an elevated pedestal (a mild stressor) was significantly blunted. Lastly, we assessed changes in basal and stress-induced c-fos and corticotropin-releasing factor (Crf) mRNA expression in brain tissue collected at 7 days withdrawal from adolescent caffeine. Adolescent caffeine consumption increased basal c-fos mRNA in the paraventricular nucleus of the hypothalamus. Adolescent caffeine consumption had no other effects on the basal or stress-induced c-fos mRNA changes. Caffeine consumption during adolescence increased basal Crf mRNA in the central nucleus of the amygdala, but no additional effects of stress or caffeine consumption were observed in other brain regions. Together these findings suggest that adolescent caffeine consumption may increase vulnerability to psychiatric disorders including anxiety-related disorders, and this vulnerability may result from dysregulation of the neuroendocrine stress response system.

Glucocorticoid Fast Feedback Inhibition of Stress-Induced ACTH Secretion in the Male Rat: Rate Independence and Stress-State Resistance.

Normal glucocorticoid secretion is critical for physiological and mental health. Glucocorticoid secretion is dynamically regulated by glucocorticoid-negative feedback; however, the mechanisms of that feedback process are poorly understood. We assessed the temporal characteristics of glucocorticoid-negative feedback in vivo using a procedure for drug infusions and serial blood collection in unanesthetized rats that produced a minimal disruption of basal ACTH plasma levels. We compared the negative feedback effectiveness present when stress onset coincides with corticosterone's (CORT) rapidly rising phase (30 sec pretreatment), high plateau phase (15 min pretreatment), or restored basal phase (60 min pretreatment) as well as effectiveness when CORT infusion occurs after the onset of stress (5 min poststress onset). CORT treatment prior to stress onset acted remarkably fast (within 30 sec) to suppress stress-induced ACTH secretion. Furthermore, fast feedback induction did not require rapid increases in CORT at the time of stress onset (hormone rate independent), and those feedback actions were relatively long lasting (≥15 min). In contrast, CORT elevation after stress onset produced limited and delayed ACTH suppression (stress state resistance). There was a parallel stress-state resistance for CORT inhibition of stress-induced Crh heteronuclear RNA in the paraventricular nucleus but not Pomc heteronuclear RNA in the anterior pituitary. CORT treatment did not suppress stress-induced prolactin secretion, suggesting that CORT feedback is restricted to the control of hypothalamic-pituitary-adrenal axis elements of a stress response. These temporal, stress-state, and system-level features of in vivo CORT feedback provide an important physiological context for ex vivo studies of molecular and cellular mechanisms of CORT-negative feedback.